TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identify their conserved interacting domains by structural analysis. We then analyse the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity is strongly reduced genome-wide in oocytes, and only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity is delayed in autosomes. These results suggest that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity.

a. Negative controls for Y2H assays for Fig. 1b b. Negative controls for Y2H assays for Fig. 1c c. Negative controls for Y2H assays for Fig. 2h.  Homo_sapiens/395-577  S  F  L V A  I S I L T  T  R  C L  L A  T  L  I  S  V N S M  G  R       e. ITC measurement of the interaction affinity between REC114 15-159 and the TOPOVIBL 559-576 peptide harbouring the W562A mutation (the wild-type control is in Fig. 1g).

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f. ITC measurement of the interaction affinity between REC114 1-159 and TOPOVIBL 452-579 (W562A) (the wild-type control is in Fig. 1g). Source data are provided as a Source Data file.   The middle sequence corresponds to the repair product by homologous recombination (HR) following CRISPR-Cas9-mediated DSB formation. The TGG codon is replaced by the GCG codon (red letters).
b. Immunostaining of RPA2, SYCP3 and H2AX in oocytes from 16 dpc wild-type (+/+),    , and Top6bl -/mice. Each data point is the mean ± SD of at least 10 sections from one ovary.
g. Testis weight in Top6bl mutants. Testis weight was measured in 2 month-old mice. Each data point is one testis (number of mice= 5, 5, 3, and 1 for wild-type (+/+), Top6bl W562A/W562A , Top6bl 17Ct /17Ct , and  c. Control for the sub-telomeric effect. Quantification was done as in Fig. 6f, but with ratios calculated between two random pools of 10 non-sub-telomeric consecutive hotspots (box plot as defined in Methods).
d. Sub-telomeric/Non-sub-telomeric hotspot density ratios for unchanged, decreased, and increased hotspots, as defined by DESeq2 analysis, for sub-telomeric regions defined using various distances from the telomeric annotated ends (1-2-3-4-5-7-10Mb). The log2 ratio was plotted and showed excess (positive values) or lack (negative values) of hotspots at sub-telomeric regions compared with non-subtelomeric regions.
e. Distribution of hotspots within 3 Mb from annotated telomeric ends and according to their DESeq2 category (unchanged, decreased, or low signal). Note the absence of increased hotspots within this subtelomeric window size. The normalized wild-type DMC1-SSDS signal is shown on the y-axis.